In the process of oxidative phosphorylation, the most enigmatic event is the mechanisms by which the energy generated by substrate oxidation is conserved and used for the synthesis of ATP. Three major types of hypotheses suggest that the redox energy is transformed into a) a proton electrochemical gradient, b) a high energy conformation of membrane components, or c) a chemical intermediate of high potential energy. Previous studies by the applicant has furnished data which are not compatible with several current models of oxidative phos phorylation in their present form. Photoaffinity labeling of mitochondria by radioactive uncouplers, and parallel equilibrium binding studies are invaluable tools in the investigation of the mechanism of uncoupling in oxidative phosphorylation. These techniques will permit the following studies; 1) Physical and chemical characterization of the uncoupler binding site, including the number and function of the proteins and the identification of the amino acids involved. 2) Purification, isolation and characterization of the protein(s) involved in uncoupler binding. This includes the elucidation of primary, secondary and possibly tertiary structure of the proteins. 3) Study of the function of uncoupler binding protein(s) by biochemical genetics and by resolution and reconstitution experiemnts. The latter involves involves isolation of the respective proteins or higher organizational units in the native state. BIBLIOGRAPHIC REFERENCES: The Role of the Mitochondiral Uncoupler Binding Site in Uncoupling of Oxidative Phosphorylation, W.G. Hanstein (1976) Biophys. J. 16, 133a. Uncoupling of Oxidative Phosphorylation, W.G. Hanstein (1976) Reviews on Bioenergetics, in press.